Ultra thin sectioning with ultra sharp diamond edge at ultra low temperature

ABSTRACT

Ultra sharp diamond edge is used for molecular and submolecular sectioning at ultra low temperature and as a high intensity point source for the emission of electrons rons and neutrons.

o v A A llmted States Patent 1191 1111 3,803,958 Fernandez-Moran A Apr; 16, 1974 ULTRA THIN SECTIONING WITH ULTRA [56] References Cited SHARP DIAMOND EDGE AT ULTRA LOW UNITED STATES A EN S TEMPERATURE 3,296,821 1/1967 Malinin 83 170 x [76] Inventor; Humberto FernandebMoran, 5807 3,495,490 2/1970 Dollhopf 83/9l5.5 x Dorchester Chicago n 2,961,908 11/1960 V1llalobos... 83/9155 X 60637 3,456,538 7/1969 Barton et al. 0. 83/171 3,236,133 2/1966 Delas 83/9155 X [22] Filed: Dec. 22, 1971 Y 7 Primary Examiner-J. M. Meister [21] 2ll135 Attorney, Agent, or Firm-Robert E. Burns; Emman- Related US. Application Data uel J. Lobato [60] Division of Ser. No. 829,267, June 2, 1969, Pat. No.

3,646,841, which is a continuation-in-part of Ser. No. [57] ABSTRACT 7 466,877, June 22, 1965, Pat. No. 3,447,366. Ultra sharp diamond edge is used for molecular and 52] U C] 83/15 83/170 83/915 5 submolecular sectioning at ultra low temperature and [51] B2621 7/08 (301m 1/06 1 as a high intensity point source for the emission of 58] electrons tons and neutrons Field of Search 83/9165, 170, 171, 15

8 Ciiiiils, IODrziviiriiFighles PATENTEBAPR 18 1914 3.803958 SHEET 1 OF 4 DIAMOND TIP (-I L- 0.0m RADIUS) TUNGSTEN OR RHENIUM HEATING' STRIP THICKNESS cA. 25-50;

MOLYBDENUM APERTURE STAINLESS STEEL OR MOLYBDENUM CATHODE CAP TUNGSTEN OR RHENIUM HEATING STRIP CERAMIC AND TUNGSTEN BASE A CERAMIC OR QUARTZ BASE TUNGSTEN OR RHENIUM POST SCREW-THREADED BASE FITTING FOR DIAMOND TIP PATENTEDAPR 16 mm a 803958 sum 3 0F 4 W. OR R. THIN STRIPS TO HIGH VACUUM PUMP LIQUID HELIUM OR LIQUID HYDROGEN LlQUlD NITROGEN CONDUCTIVE COATING DIAMOND KNIFE EDGE (TIP RADIUS) Co 0.0l TO 0.00! MICRON FLUORESCENT SCRE EN PATENTEUAPR 16 1914 SHEET E [If 4 ULTRA THIN SECTIONING WITH ULTRA SHARP DIAMOND EDGE AT ULTRA LOW TEMPERATURE This application is a division of my application Ser. No. 829,267 filed June 2, 1969, now U.S. Pat. No.

' 3,646,841, which in turn was a continuation-in-part of application Ser. No. 466,877, filed June 22, 1965, now

U.S. Pat. No. 3,447,366.

It is widely recognized that ultra sharp diamond knives and associated ultramicrotomy apparatus of the type developed and systematicallyintroduced in research laboratories and in industry by the applicanthave considerably advanced the state of prior art in the preparation of very thin slices of materials, and in ultraprecision machining. His pioneering work in this specialized field has resulted in the production of the highest quality metal surfaces presently attainable with finishes and tolerances in the low-microinch range.

This unprecedented, readily attainable microinch accuracy is now achieved directly by continuous turning operation using the diamond knife, without the traditional time-consuming and very costly additional work of lapping, buffing or polishing.

The cutting-edge sharpness radius of the order of 0.10 microinch, and the regularity of the diamond knife cutting edge are of decisive importance for this purpose, as compared with the measured cutting-edge radius of well-sharpened conventional tools which is about 300 microinches. As shown by careful studies, this far greater cutting-edge radius and irregularity of the best standard tools results in the production of high frictional forces on the cutting edge which generates a poor surface finish by tearing and deforming the material plus accelerated wear and abrasion of the edge.

However, with a diamond cutting tool having an edge thickness of 0.001 to 0.01 micron results are consistently obtained that practically approach the idealized condition for optimum sectioning. Since the zone of compressive yielding and flow around the edge is reduced to a vertical length of a few microinches, more uniform flow and yielding of the material at the active tool edge is achieved with consequent improvement in the surface finish of thecut. Moreover, very precise sizing can be maintained because cut depths of only 5 to microinches are readily made.

It is therefore essential to determine accurately the dimensions and properties of these ultra sharp cutting edges as an indispensable factorin the production and testing of the diamond knife, consideredto be the most sophisticated cutting tool available today.

Prior to my workin this field, there were'no adequate methods for direct imaging andmeasuring the cuttingedge sharpness radius of dimensions below the resolving power of the light microscope (0.2 to 0.4 micron). In fact, the active cutting region of our sharpest cutting tools remained invisible until suitable methods were devised which permitted successful application of the hundredfold higher resolving power of'the electron microscope for direct observation of these submicroscopic structures. Direct visualization and precise measurement of the ultra sharp cutting edges was an integral part of research program in ultramicrotomy, and played a key role in the development of the diamond knife. The critical measurement and testing procedures for the cutting edges which have been developed in the course of this work represent the basis for a rational approach to the complex problems encountered in the new domain of ultraprecision machining and molecular sectioning.

Conversely, the possibility of working with stable cutting edges of submicroscopic dimensions, and of studying their interaction with selected specimens under controled conditions'has opened up a new field of investigation, disclosing new phenomena which are operative predominantly at the level of molecular organization.

Thus, for example, with a diamond cutting-edge sharpness radius measuring only 10 to 100 A., which corresponds to some six to 60 carbon atoms strongly covalently bonded, it is actually possible to section or cut up certain long-chain organic polymers into their constituent chemical subunits of molecular dimensions. By using the special ultramicrotome apparatus described earlier operating at very low temperatures it has been possible to cut starch macromolecules into their constituent sugar molecules endowed with quite different chemical properties.

This controlled modification of molecular'structures represents a new form of manipulation, extending well beyond the established definition of mechanical sectioning, and leading into the realm of a novel modality of preparative chemistry and physical chemistry performed under conditions of mimimum perturbation in a low entropy environment.

The methods described in this application were used primarily in connection with the production and testing of diamond knives; since diamond is the hardest naturally occurring material, and its crystalline structure makes it possible to reproducibly obtain stable cutting edges of molecular dimensions, using appropriate techniques and apparatus, of the type described in my U.S. Pat. Nos. 2,961,908, 3,060,781, and 3,447,366.

However, the measurement and testing procedures can likewise be applied to determine the sharpness and properties of the active cutting edges of all kinds of cutting tools, including glass knives for ultramicrotomy, razor blades and related steel cutting edges, the wide variety of well-sharpened machine tools, and in general all cutting tools used in science, industry and technology where optimum sharpness and regularity are essential. It should be pointed out that accurate determination of cutting-edge sharpness radius and related properties of active cutting tools is of fundamental significance as an integral part of any manufacturing or production procedure designed to systematically achieve efficient cutting tools forsectioning all types of materials, in a controlled and economic way. This difficult field has been neglected in the past, partly owing to technical limitations inherent in light microscopy. However, now that ultraprecision machining and related disciplines are assuming vital importance in an era of increased automation, which depends for its success on absolute uniformity of operation and reproducibility of the finished product, the refined measurement techniques described here are of key operational value. In many ways a similar relationship can be invoked between the improvements in measuring techniques and the subsequent spectacular advances in ultrahigh vacuum technology. It was only after reliable devices and techniques for measuring ultra-high vacuum, like the Alpert gauge, became available that the whole field of attaining and monitoring ultrahigh vacuum environments came into being and could be systematically developed on a rational basis.

The invention relates generally to measurement and testing procedures for ultra sharp cutting edges. The

separate steps can be carried out individually in different stages, but are best applied sequentially in systematic combination, since they are mutually supplementary, and finally yield an integrated picture of the cutting edge configuration and properties.

The procedure embodies the following distinguishing features incorporated in the successive operational stages:

a. Direct examination of the ultra sharp cutting edge and its adjacent components, carried out step by step continuously at the different levels of magnification extending from the range of light microscopy to electron microscopy, electron diffraction and related electron optical techniques, in a non-destructive, highly accurate, and reproducible quantitative analytical way;

b. Supplementary examination of the surface characteristics of the cutting edge facettes and relationship to the active cutting radius of electron optical examination of a special type of high resolution indentation replica especially developed for this purpose;

0. Ultimate critical test of the performance and stability of ultrasharp cutting edges by actual cutting of ultrathin serial sections of selected specimens, such as metals, hard crystalline substances, macromolecular materials and biological specimens with a highly ordered internal structure. The quality and properties of the cutting edge can be accurately evaluated by combined electron microscopy, electron diffraction, physical, and physical-chemical studies of the resulting ultrathin sections and the exposed speciment block surface;

d. In special cases, and particularly for routine monitoring and inspection of large numbers of cutting tools with ultra sharp edges,'certain procedures for examinations based on the characteristic electron optical, electrical, and ion-optical properties of these cutting edges can be adapted for automated inspection on an assembly line basis.

In the following description of the apparatus end method in accordance with my invention, reference is made to the accompanying drawings in which:

FIG. '1 is a schematic perspective illustration of a diamond tip point cathode source;

FIGS. 1A, 1B and 1C are enlarged schematic views of the diamond tip point cathode source and associated parts of FIG. 1;

FIG. 2 is a characteristic brightness curve of the point source illustrated in FIG. 1;

FIG. 3 is a schematic sectional view of a high intensity field-emission electron and ion sources using an ultra sharp diamond edge; and

FIGS. 3A and 3B are enlarged schematic views of the ion source shown in FIG. 3;

FIG. 4 is a schematic perspective view of apparatus for molecular and submolecular sectioning.

FIG. 4A is an exploded schematic view of a cryogenic motor of the apparatus shown in FIG. 4.

a. Direct examination of the ultra sharp cutting-edge by high resolution light microscopy and electron mi croscopy, and related electron-optical techniques.

This procedure can best be illustrated by referring to a typical example such as a diamond knife of the type described in my U.S. Pat. No. 3,060,781. A representative diamond knife comprises a diamond body portion with a perfectly uniform and stable cutting edge of about 3 to 7 mm length, and with a cutting-edge sharpness radius of about 10 to I00 A. units (0.00] to 0.01 micron). These diamond knives usually have facettes defining edge angles of about 40 to 50 for cutting plastic materials, and of to for cutting metals and other hard substances. However, contrary to certain claims, these edge angles do not define the active cutting-edge of the knife. Instead, the active cutting region is several hundred Angstrom units from the edge line, since it is built up of the regularly disposed crystalline unit layers of the diamond arranged in stable configuration to give smooth facets. In order to carry out the first series of optical tests the diamond knife embedded in its metal support must be mounted in a specially designed holder which permits it to be very accurately positioned and oriented at preselected optimum angles for direct examination by light microscopy, and subsequently by electron microscopy. A photomicrograph of the edge taken at the highest light microscope magnifications (1,000x to 2,000x) shows a perfectly regular, straight and extremely smooth cutting edge facet. The actual cutting edge is not directly visible, since it lies beyond the resolving power of the ordinary light microscope. However, when examined under optimum conditions with point light sources of very high intensity (e.g., Xenon lamps, lasers, etc.) with the dark field ultramicroscope the active cutting edge can be faintly detected by virtue of its light-scattering properties. This allows detection of small cracks and irregularities in the edge, but still does not permit direct imaging of the criticalactive cutting edge. Nevertheless, improved light microscopy techniques such as interference microscopy permit accurate determination of the general quality of the cutting edge and adjoining facet. This is especially useful when comparing the uniform contours and regular spacing of the drak interference bands indicating a perfectly plane and smooth diamond edge and facet, with the curving and irregularly distorted interference fringes corresponding to the submicroscopic irregularities of a polished steel razor edge. Following these initial observations which can be readily carried out routinely during the various steps of the final polishing of the diamond or other type of ultrasharp cutting edge, the decisive electron optical examination is carried out.

For this purpose the same holder can be used, suitably adapted for introduction into the high vacuum specimen chamber of an electron microscope. These studies require the use of a specially modified high resolution electron microscope operating at accelerating voltages of 40 to kV in most cases (also in the low voltage range of 5 to 10 kV for certain applications), provided with a special pointed filament source with single-crystal tungsten tips of the type developed by the applicant. By means of these pointed sources highly coherent microbeam illumination (beam diameters ranging from 100 A. to 10 microns) of low energy spread and very low intensity can be used, in conjunction with suitable high or ultrahigh vacuum specimen chambers preferably cooled with liquid nitrogen or hydrogen (vacuum of the order of 10 to 10 mm Hg) to prevent the deleterious contamination of the knife edge during illumination with the electron beam. Under these optimum conditions the properly oriented cutting edge can be very clearly seen directly and photographed at electron optical magnifications ranging from 2,000x to about 200,000x, with subsequent photographic enlargement to attain total magnifications of half a million to about one million diameters. With a focused electron microbeam of adequately low intensity to avoid specimen irradiation damage the electron microscope can readily be employed to produce a distinct shadow image of the diamond knife edge and supporting region, at magnifications adequate to resolve fine details of the edge in order to a few hundred Angstroms. Imaging by reflection electron microscopy can also be readily accomplished by using oblique illumination and tilting the knife so that the incident electron beam makes a very small angle with its surface. This simple technique yields valuable data on the surface structure of the knife facet and of the active cutting edge, supplementing the results obtained by examination of the surface replicas.

However, the most important information is obtained by straightforward transmission electron microscopy with microbeam illumination at high magnifications, essentially because the active ultra sharp cutting-edge is ultrathin, and therefore it alone is electron transparent and can be selectively imaged. This remarkable ef-' feet which was discovered in the course of these studies has provided to be of extraordinary value as one of the most direct and powerful means of studying ultra sharp cutting edges at the highest electron-optical magnifications. It is readily understandable, since the ultra sharp cutting edges with a, radius of merely to 100 A are built up of a relatively small number of the crystalline unit layers of diamond to form anzultrathin wedgeshaped structure which is at most only a few'hundred Angstrom units thick at the base, and therefore stands out selectively as an electron transparent window against the rest of the knife base which is electronopaque. The fact that the ultrathin active cutting-edge of an ultra sharp cutting tool is essentially an electrontransparent window is of unique operational value because it permits us to study directly all structural details clearly visible in this most important region under excellent conditions which minimize specimen radiation damage. This effect is also clearly discernible when using electron diffraction techniques, since there is a well-defined transition from a reflection-type electron diffraction pattern to a transmission electron diffraction pattern precisely along the boundaries of the ultrathin cutting-edge. Although these characteristic electron-optical effects are most clearly recorded in diamond knife cutting-edges, they have also been observed under specially controlled conditions in other types of ultra sharp cutting edges in glass and steel razor edges, as well as other kinds of related cutting tools. This ultrasharp knife cutting-edge electron transmission effect can serve as a basis for quantitative studies and selective detection of all types of cutting edges of these dimensions. After suitable calibration of the transmission electron microscope image or electron diffraction pattern using single-crystal graphite or epitaxially grown thin films asa standard for checking the thickness and related structural parameters, a conventional electron microscope can be readily adapted to serve as a routine monitoring and detecting device for these cutting edges of molecular thickness.

Other similar types of molecular cutting-edge detectors" based on the selective electron-transmission affect are suitable for automated routine inspection and control of ultra sharp cutting edges on an assembly line basis. Some of these devices, which will be the subject of a separate application, are considerably simplified by making use ofa collimated electron source (e.g., radioisotope ,B-emitter sources) suitably arranged so that the electrons selectively transmitted through the ultrathin cutting-edge are then recorded on a sensitive indicator, such as an image intensifier or photographic emulsion.

b. Examination of ultra sharp cutting-edges by high resolution replica techniques.

Ordinary replica techniques which provide an indirect view of a surface mold or impression on thin plastic and metal films are very difficult to apply to the study of ultra sharp cutting edges, because the active cutting edge is largely obliterated or can actually be damaged during the required stripping process, and there is considerable distortion of the initial structural relationships. It was therefore necessary to develop a simple and reliable replica technique which would yield an accurate impression of the active cutting edge and adjoining knife facet suitable for examination by high resolution electron microscopy. Among the numerous variants that were developed the following high resolution indentation-replica technique applied preferably on thin films was found to be the most useful method for accurately determining the cutting-edge sharpness radius, and the surface detail of the adjoining knife facets, including the orientation and size of the abrasive particle tracks in the final polishing process of the ultra sharp diamond knife edges.

It is based on the precise indentation track of the active cutting edge which is produced by perpendicular indentation of the knife on ultrathin (about 50 to 200 A.) carbon-plastic films floating on a liquid surface or supported on a smooth soft support. The atomically smooth and perfectly plane films are produced by-highvacuum evaporation of carbon on a freshly cleaved mica surface, followed by coating with a collodion or Formvar film. The composite films are then stripped off from the mica by simple floating on water, or glycerol, or on gelatin or plastic substrates. The diamond knife or other type of ultra sharp cutting tool is mounted on a special holder connected with a sensitive balance to permit controlled ultra-low-load identation of the smooth surface film, when the edge descends perpendicular on this ultrathin surface. The resulting indents (of which several can be made in rapid succession without damaging the sensitive knife edge) are readily visible with phase-contrast or dark-field microscopy. However, the actual active cutting edge can only be seen by direct examination of the replica (with subsequent carbon or platinum shadow-casting if necessary) by high resolution electron microscopy. The cutting-edge sharpness radius stands out clearly and can be accurately measured, allowing for the contribution of the shadowing material. Adjacent to this cutting edge replica, the individual crystalline unit layers of the diamond facet can be clearly discerned. Profiles of the cutting edge can also be studied by means of stereoscopic pairs recorded with a special tilting state in the electron microscope. The replica technique can be further refined by carrying out the whole procedure in an ultrahigh vacuum chamber (10 to 10" mm Hg) indenting a freshly prepared ultrathin carbon film evaporated directly on a cooled, clean glycerol surface, followed immediately by carbon or carbon-platinum shadow casting of the resulting indents of the ultra sharp cutting edges.

Large number of measurements performed on these replicas clearly show that the active cutting-edge sharpness radius of diamond knives are exceptionally regular with dimesions of the order of 10 to 30 A. in the best knives. Moreover, these active cutting edges are remarkably stable after prolonged used, as determined by combined application of the replica and the other measurement techniques described here.

There variants of the replication techniques include direct evaporation of NaCl-carbon films on to the suitably oriented diamond or other ultra sharp cutting edges in an ultrahigh vacuum, followed by stripping off in an ultrafiltered water surface. Shadowgrams of the ultra sharp cutting-edges can also be obtained by collimated heavy metal shadow-casting of the cutting tools resting on a freshly cleaned mica surface.

c. Determination of properties and stability of ultra sharp cutting edges by cutting of ultrathin serial sections of metals and selected specimens, followed by examination of the sections and surface of the specimen block.

All of the previously described methods give valuable data on the dimensions and configuration of the ultra sharp cutting edges, but the most critical test for their stability and performance can only be provided by actual ultrathin sectioning. For this purpose a special instrument, such as the ultramicrotome described in my US. Pat. Nos. 2,961,908 and 3,091,144, preferably operating at very low temperatures, is employed. Metal specimens, such as single-crystals of aluminum, copper, gold, zinc, or platinum with a shaped pyramid or cone" tip, preferably only 10 to 100 microns in diameter, are mounted on the specimen holder of the very accurate ultramicrotome with a continuously adjustable feed, and a measured reproducibility. 0. Determination of properties and stability of ultra sharp cutting edges by cutting of ultrathin serial sections of metals and selected specimens, followd by examination of the sections and surface of the speciment block.

All of the previously described methods give valuable data on the dimensions and configuration of the ultrasharp cutting edges, but the most critical test for their stability and performance can only be provided by actual ultrathin sectioning. For this purpose a special instrument, such as the ultramicrotome described in my US. Pat. Nos. 2,961,908, and 3,091,144, preferably operating at very low temperatures, is employed. Metal specimens, such as single-crystals of aluminum, copper, gold, zinc, or platinum with a shaped pyramid or cone" tip, preferably only 10 to 100 microns in diameter, are mounted on the specimen holder of the very accurate ultramicrotome with a continuously adjustable feed, and a measured reproductibility without the specimen of about i 10 A. After careful adjustment of the optimum cutting angle of the ultra sharp (diamond) knife, keeping the clearance angle as small as possible (preferably less than 1) the thermal specimen advance is activated, and preferably uniform serial sections are cut and collected in the form ofa tenuous ribbon floating on the water (or other suitable liquid surface for low-temperature work) contained in the trough. Metal sections are ideal test specimens for'the ultra sharp knives, because in contrast to the usual plasticembedded specimens which have a tendency to spread-out" sectioning, sectionig, the metal sections retain their original dimensions, and give a faithful reproduction of the diamond cutting edges, glass and steel knives cannot be used for ultrathin sectioning of hard materials, such as germanium. It is important to bear in mind that the process of cutting a thin metal section is accompanied by a rather severe shear deformation of the section and a momentary rise in temperature, which can be as high as 300 C when sectioning is performed at room temperature. However, when'ultrathin sectioning of metals is carried out at temperatures below about 1 C this deleterious temperature rise can be largely avoided (because of the typical high termal conductivity, low thermal expansion and low compressibility of diamond).

The resulting ultrathin sections of metals are about 50 to 200 A., as determined by combined electron microscopy and electron diffraction studies, using control films and crystalline lamellae of known thickness for accurate calibration. The structure of the metal sections is a very sensitive indicator of the dimensions and quality of the ultra sharp cutting edges, since a blunt" knife will result in section failure, edge deflection will result in irregular and compressed sections, etc. The corresponding block surface from which the sections have been cut exhibits an exceptional degree of finish (in the microinch range) which is superior to that obtained by any of the known mechanical polishing procedures. Nevertheless, even slight imperfections in the cutting edge show up as minute tracks in both sections and block surface.

One of the most sensitive criteria for determination of edge thickness and quality is provided by the preparation of ultrathin serial sections of macromolecular components and highly ordered biological tissues with a precise built-in period which serves as an internal calibration for the section thickness. Thus, an ultrathin section of about thickness as revealed in an electron micrograph of the highly regular paracrystalline lattice of insect wing inclusions depicting individual protein molecules with a regular spacing of 80 to 90 A. It is actually possible to section a virus particle, like the tobacco mosaic virus, of molecular dimensions into two or more than 80 A.

Finally, by using the best ultra sharp cutting edges and low-temperature ultramicrotomy, it has been possible to produce serial sections having molecular or submolecular thickness, prepared from frozen macromolecular substances. A native crystal of the enzyme catalase with a regular spacing of 90 A. has been cut into sections which are about A'. thick; another catalase crystal has been sectioned into slices of about 45 A. thickness. This can only be directly visualized by high resolution electron microscopy when working with extremely thin sections of these molecular dimensions.

An even more sensitive criterion for cutting tool sharpness can be obtained by sectioning long-chain polymers such as starch into their constituent sugar molecules, as mentioned previously. This true molecular sectioning" results in characteristic chemical changes (such as the different specific chemical reactions of sugar molecules, prepared by controlled breaking up of the starch molecules) which can be correlated with the known structural parameters of these molecules, and in turn related to the cutting-edge sharpness of the knife.

It is important to emphasize that there is a very close correlation and excellent agreement between the results of the different methods described here for determination of the dimensions and properties of these ultra sharp cutting edges.

Apparatus for effecting ultramicrotomyat low temperatures to produce serial sections of long-chain polymers and other important macromolecules into their active constituent components is illustrated in FIG. 4. Because the diamond knife used in conjunction with the cryo-ultra-microtome, as shown in FIG. 4, can cut specimens as thin as A. to 100 A., it is now possible to literally effect chemical changes by cutting. Thus, by means of molecular sectioning a starch molecule can be cut up in such a way that it becomes sugar. More significantly, it is now possible to cut the master molecule DNA into viable segments which can be subsequently spliced onto other DNA molecules.

In the apparatus shown in FIG. 4, a'diamond knife 1 of an edge thickness 0.001 to 0.01 micron is attached to a special holder 3, provided with adjusting screws 2 and can be cooled down to temperatures of about 1 to 30 above absolute zero by cryogenic fluid supplied through a tube 4 to a cavity in the holder to provide a bath for picking up the ultrathin serial sections. The holder can be precisely adjusted by a micrometer 5. Lateral movements of the holder can also be controlled by a screw 6.-A flexible cooling strip 7, for example copper, ensures the required low temperature which is critical for biological specimens. The entire assembly base 8 is filled with a cryogenic fluid such as liquid nitrogen or helium through a flexible tube.9. The specimen-10 is affixed to a holder 11 which is also cooled through a device 12 and is attached to a highly precise cylindrical metal rotor 14 which is kept cold through special cooling coils 13, 15. A conventional motor drive operating in a high vacuum can also be attached to the rotor at 13, 15. Special diamond or equivalent point bearings 20 maintain the constancy of the rotor ends 16. Provisions are made by means of copper or other cooling strips 17, 18 to keep the bearings at the required low temperature. The bearing holders 20, 21 and 22 are needed for extra stability. The entire ultramicrotome is preferably enclosed in a high vacuum enclosure 24 with seals 23. An ultrahigh vacuum can be maintained in this enclosure by using an ion pump 25 in conjunction with cryogenic pumping. Since the system is very sensitive to temperature changes, radiation shields 27 are provided. The rotor can also be driven by a special cryogenic magnetic motor drive 29 comprising pick off pins'A, an upper bearing B, a stator shell C, rotor'D and lower bearing E. The entire assembly is mounted on a vibration proof case 26.,

With the cooling means illustrated and described, it is possible to cool the specimen and its holder and the diamond knife and its holder and also the whole environment of the specimen and knife down to a temperature of the order of 0.00l above absolute zero. By cutting with a diamond knife having an edgethickness of 0.001 to 0.01 micron and by carrying out the cutting at a temperature at orbelow 20 K (the temperature of liquid hydrogen) it is possible to obtainmuch'thinner cuts than heretofore possible. Preferably the cutting is effected at a temperature below 50 K. While liquid heliurh provides a temperature of 4.2 K, it has been effect accurate initial positioning ofthe diamond knife relative tothe specimen carried by the holder 11 on the rotor. M ans is incorporated in the mounting of the specimen holder for moving the specimen toward the knife holder by minute increments to cut successive ultrathin slices of the specimen as the rotor rotates. Such means may utilize the thermal expansion of a metal member, for example a member of INVAR alloy. The thermally expansive member may be sufficiently heated by light shining on it. Preferably, however, movement of the specimen is effected by magnetostrictive means. Thus, the specimen mounting may comprise a bar or other member of the rare earth dysprosium which deforms physically in response to a magnetic effect.

The described cryogenic ultramicrotome can be scaled down (as shown in the scale) from 50 to l centimeter approximately. The molecular or pauciatomic method has beenthorougly tested and can be used to section any type of material ranging from biological specimens to metals, catalysts etc. This type of bio engineering which has been made possible by the introduction of the diamond knife, has numerous other applications extending into the micro electronic industry (e.g., production of micro coils and micro transformers or capacitors by precision sliding). The key feature of this approach is given by the capabilities of the electron microscope which not only make it possible to see directly the critical domain of atoms and molecules but also to be able to design matter in this pivotal range which was hitherto inaccessible.

d. Examination of ultra sharp edges by ion-optical, field emission and electrical measurement techniques.

One of the most interesting analytical approaches in the study of ultra sharp cutting edges, particularly of diamond, which is being actively investigated by the applicant, derives from the application of pointprojection microscopes. This type of microscope, as first described by E. Mueller, consists of a sharp metal cathode (e.g., very fine tungsten point with a radius of onlya few hundred Angstroms) located; in a highly evacuated or gas-filled tube with the fluorescent screen and an interposed anode ring at a specified distance. With a sufficiently high potential (usually of 10 to 20 kV) between anode and point, electrons or ionized helium atoms will be drawin from the point and imaged directly on the screen displaying the atomic structure of the point at magnifications of several million. The magnification can be readily calculated as a function of the radius of the cathode .and the distance to the screen. A diamond ultrasharp edge of 10 to A. which is coated with tungsten or platinum by high vacuum evaporation can be actually considered as an extended'point source in such a point projection microscope. Experiments demonstrate in fact that such a diamond knife edge is an excellent point source which gives exceptionally bright images both in the field emission and in the field ion microscope. From these images the dimensions of the emitting area which correspond to the active cutting edge of the knife can be determined. Moreover, as shown by the classic work of E. Mueller, this technique permits in principle a very accurate determination of the atomic structure of these emitting tips, investigation of crystalline defects and other important properties.

Systematic work in this field combined with investigation of the characteristic elecron-optical and electrical field properties exhibited by ultra sharp stable edges of these dimensions are of great potential value for atomated inspection of ultra sharp cutting edges of all kinds.

A new type of source for electrons, ions, X-rays, neutrons of controlled exceptionally high intensity and coherence is illustrated in FIG. 1.

A diamond point or edge 30 having a tip radius of approximately 0.001 to 1 micron or up to about microns and of a precisely determined shape acts a permanent substrate for sources of a suitable material such. as tungsten, rhenium, lanthanum, barium, caesium and other related materials of suitable work function and physical properties for electron emission. As shown in drawing FIG. 1, the diamong tip or edge is accurately mounted on a suitable ceramic or tungsten base 31 which is mounted on a ceramic base plate 32 so it can be precisely aligned. For example the base 31 is provided with a reduced threaded portion 31a which extends through an upper washer 33, a hole in the base plate 32 and a lower washer 34 and is secured by a nut 35. The hole in the base plate is sufficiently large to permit adjustment of the base 31 carrying the diamond tip whereupon the nut 35 is tightened to hold the diamond tip in adjusted position.

Associated with the diamond tip 30 is a heater and emitter 36 in the form of a strip or coil or other suitable configuration. The emitter-heater is shown by way of example as a strip extending between two posts 37 and having a hole through which the diamond tip extends. The strip 36 and also the posts 37 are preferably of tungsten or rhenium. The tungsten or rhenium strip 36 is heated to a high temperature by current supplied through the posts 37 in order to evaporate a thin film of this emitting material onto the diamond point.

The assembly is placed in a cathode gun (see FIG. 1) of suitable shape and material and serves as an electron source which, in conjunction with an anode (not shown in drawing) is used to accelerate an electron beam of very high brightness, for example, 10 to 10 amperes cm Sterad as shown in FIG. 2. As illustrfated in FIG. 1, the gun includes a cathode cap 38 of stainless steel or molybdenum having a molybdenum member 29 with an aperture through which the tip of the diamond projects. The aperture is of a size to receive the diamond tip closely without touching it and may, for example, be of the order of 0.1 to 1 mm. The cap shields the anode from emission by the strip 36 and thereby assures that only the diamond tip emits. A bias voltage of, for example, 1001,000 volts is applied between the emitter 36 and the cap 39 while an accelerating voltage .of, for example, 1,000 to 100,000 volts is applied between the cap and anode.

In order to increase the life of the cathode, a reservoir of the emitter is provided. This may, for example be in the form of emitter material preferably in paste forms introduced into a transverse hole 30a in the diamond tip or a ring of said material around the base of the tip as illustrated at 30b. The reserve material gradually migrates to the hotter tip as the emitter material on the tip is used up.

The characteristic beam current obtained with a cathode according to the invention in conventional high vacuum tubes, for example 10" to 10 mm Hg, used in electron microscopes and in television tubes and related devices is orders of magnitude'higher than the normal thermionic sources. The unique advantages of this type of source are (a) permanent critical alignment since the diamond which acts as a substrate is not affected and the source can be readily replenished simply by evaporating a new film onto the diamond tip. Since diamond has the lowest thermal expansion coefficient and is the hardes substance known, it will withstand the great stresses normally involved. The expected life of cathodes in accordance with the invention is months or years, as compared with the average lifetime of the usual cathodes measured in several hundred hours only. This source can be applied to replace the existing cathode sources for electron microscopes, television tubes, X-ray tubes, etc. without appreciable modifications. In view of its exceptional stability and precise alignment, for example in an electron microscope where alignment has to be adjusted every time the tungsten filament source is replaced, it is particularly suitable for applications such as space probes, etc. requiring months and years of unattended use. The diamond tip may be a point or an edge according to the application of the cathode.

The thin film of emitter material on a diamond tip substrate in accordance with the invention permits critical centering and height adjustment of the emitter relative to the cap to assure stable bias voltage and emission control. This electrode and cap design is applicable to both cathode and anode structures, combining optimum micro-geometry and operational conditions to obtain space-charge-free emission and significant field enhancement. The emitter in accordance with the invention may operate as a thermionic emitter or as a temperature-field emitter.

A similar type of source as shown in FIG. 3 is used in a field emission electron and ion tube using either a diamond point or a diamond knife edge 40 with a tip radius of about 0.01 to 0.001 micron serving as the substrate for accurately deposited tungsten or rhenium thin film strips, and working in an ultra high vacuum, for example of the order of 10 to 10 mm Hg. This type of source becomes the equivalent of a comb type source representing many hundreds or thousands of individual field emission point sources.

Alternatively, the diamond tip may have a continuous coating. The tip 40 is mounted on a holder 41 shown in the form of a rod extending down through a vessel 42 cooled by a cryogenic fluid, for example liquid hydrogen or helium. The diamond tip 40 is surrounded by an emitter Ch of a suitable emitter material, for example tungsten or rhenium. The point of the diamond tip extends through a hole in a grid cap K, for example of molybdenum. A second grip cap W having a larger aperture is interposed between the diamond tip and an anode A.

The cathode-anode assembly is surrounded by an annular chamber 43 which is likewise cooled by a cryogenic fluid, for example liquid nitrogen, and is enclosed in an envelope in the form of a tube 44 in which a high vacuum is maintained. Shielding is provided by conductive coatings 45 and 46 connected to ground. A flourescent tube 47 is provided at the end of this tube. It has been demonstrated that with an emitter of this kind one can obtain enormous current densities, for example, of the order of 10 amp/cm, thus exceeding other conventional cathodes by a factor of a million. For example, by using this type of source in the T-F mode (temperature and electrical field emission) with accelerating voltages of about 10,000 to 200,000 or more, these point shaped cathodes operating in parallel can yield peak powers of 3MW or more corresponding to energies of to 10 ergs or more in microsecond bursts. Further modifications of this type of source for producint high energy and highly coherent electron beams, X-rays, neutrons, etc. may be made.

It will be understood that features of the embodiments illustrated in the drawings and herein described are mutually interchangeable in so far as they are compatible and that many modifications in size, materials and specific configuration and design may be made.

What I claim is:

l. A method of making ultra-thin serial sections of a specimen, comprising the steps of mounting said specimen on a first holder in a chamber, mounting on a second holder in said chamber a diamond knife having a cutting edge thickness of the order of 0.001 to 0.01 microns, drawing an ultra high vacuum in said chamber, cooling said chamber, first and second holders, specimen and knife to a temperature lower than 5 K, moving said knife and specimen relative in a first direction relative to one another to make a first cut of said specimen with said knife, moving said knife and specimen relatively toward one another in a second direction transverse to'said first direction a distance of 10A. to l00A., again moving said knife and specimen relative to one another in said first direction to make a second cut of said specimen parallel to the first cut, thereby cutting a section of said specimen having a thickness of 10A. to 100A. corresponding to therelative movement of said knife and specimen in said second direction, re-

peating said movements alternately in said first and second directions to cut a series of sections of said specimen and collecting said sections in a bath of cryogenic 2. A method according to claim 1, in which said chamber, first and second holders, specimen and knife are cooled with liquid helium and in which said bath is liquid helium.

3. A method according to claim 1, in which one of said holders comprises a thermally expansive member, and in which light is directed on said member to cause expansion thereof to effect relative movement of said knife and specimen in said second direction.

4. A method according to claim 1, in which one of said holders comprises a magnetostrictive means, and in which a magnetic effect is applied to said magnetostrictive means to effect relative movement of said knife and specimen in said second direction.

5. A method according to claim 1, comprising shielding said holders, specimen and knife from radiation effecting temperature changes.

6. A method according to claim 1, comprising cooling said holders by thermal conduction through flexible thermal conductors.

7. A method according to claim 1, in which said specimen comprises macromolecules, comprising cutting said macromolecules into constituent components.

8. A method according to claim 1, further comprising isolating said first and second holders from external vibration. 

1. A method of making ultra-thin serial sections of a specimen, comprising the steps of mounting said specimen on a first holder in a chamber, mounting on a second holder in said chamber a diamond knife having a cutting edge thickness of the order of 0.001 to 0.01 microns, drawing an ultra high vacuum in said chamber, cooling said chamber, first and second holders, specimen and knife to a temperature lower than 5* K, moving said knife and specimen relative in a first direction relative to one another to make a first cut of said specimen with said knife, moving said knife and specimen relatively toward one another in a second direction transverse to said first direction a distance of 10A. to 100A., again moving said knife and specimen relative to one another in said first direction to make a second cut of said specimen parallel to the first cut, thereby cutting a section of said specimen having a thickness of 10A. to 100A. corresponding to the relative movement of said knife and specimen in said second direction, repeating said movements alternately in said first and second directions to cut a series of sections of said specimen and collecting said sections in a bath of cryogenic fluid.
 2. A method according to claim 1, in which said chamber, first and second holders, specimen and knife are cooled with liquid helium and in which said bath is liquid helium.
 3. A method according to claim 1, in which one of said holders comprises a thermally expansive member, and in which light is directed on said member to cause expansion thereof to effect relative movement of said knife and specimen in said second direction.
 4. A method according to claim 1, in which one of said holders comprises a magnetostrictive means, and in which a magnetic effect is applied to said magnetostrictive means to effect relative movement of said knife and specimen in said second direction.
 5. A method according to claim 1, comprising shielding said holders, specimen and knife from radiation effecting temperature changes.
 6. A method according to claim 1, comprising cooling said holders by thermal conduction through flexible thermal conductors.
 7. A method according to claim 1, in which said specimen comprises macromolecules, comprising cutting said macromolecules into constituent components.
 8. A method according to claim 1, further comprising isolating said first and second holders from external vibration. 